STRUCTURAL ASPECTS AND IMMUNOLOCALIZATION OF THE F-420-REDUCING AND NON-F-420-REDUCING HYDROGENASES FROM METHANOBACTERIUM-THERMOAUTOTROPHICUM MARBURG

The F-420-reducing hydrogenase and the non-F-420-reducing hydrogenase (EC 1.12.99.1.) were isolated from a crude extract of Methanobacterium thermoautotrophicum Marburg. Electron microscopy of the negatively st ained F-420-reducing hydrogenase revealed that the enzyme is a complex with a diameter of 15.6 nm. It consists of two ring-like, stacked, pa rallel layers each composed of three major protein masses arranged in rotational symmetry. Each of these masses appeared to be subdivided in to smaller protein masses. Electron microscopy df negatively stained s amples taken from intermediate steps of the purification process revea led the presence of enzyme particles bound to inside-out membrane vesi cles. Linker particles of 10 to 10 kDa which mediate the attachment of the hydrogenase to the cytoplasmic membrane were seen. Immunogold lab elling confirmed that the F-420-reducing hydrogenase is a membrane-bou nd enzyme. Electron microscopy of the negatively stained purified non- F-420-reducing hydrogenase revealed that the enzyme is composed of thr ee subunits exhibiting different diameters (5, 4, and 2 to 3 nm). Acco rding to immunogold labelling experiments, approximately 70% of the no n-F-420-reducing hydrogenase protein molecules were located at the cel l periphery; the remaining 30% were cytoplasmic. No linker particles w ere observed for this enzyme.